This proposal describes the synthesis and validation of `smart' ultrasound contrast agents that become active in response to interaction with localized biochemicals for non-invasive, accurate diagnosis and staging of breast tumors. Biopsy and histology are often performed to investigate suspicious lesions found in a routine mammogram because these procedures provide the most relevant information for diagnosis and staging of tumors. However, biopsies are invasive, costly and potentially dangerous. The development of a non-invasive tool that could reliably and inexpensively image tumors would help to reduce the number of unnecessary biopsies for breast cancer patients and the associated physical, financial, and psychological costs. In this proposal, we seek to develop high-resolution ultrasound imaging methods to identify accurately if suspicious lesions in mammograms are worth studying or treating further, as well as screen for potential drug resistance. Ultrasound has been successful for contrast-enhanced blood pool imaging, but the contrast agents often required to improve image resolution are unable to accumulate within the extravascular space owing to their microscale sizes, thus preventing the staging of tumors. Better tumor penetration can be achieved by utilizing sub-micron contrast agents; unfortunately, these cannot provide high contrast due to lower acoustic scattering cross-sections and potential instability. The balance between size and response to ultrasound can be resolved by using responsive contrast agents that become only active after uptaken by the target cancer cells. In such a scheme, the background signal coming from the contrast agents that are still in the blood stream can be eliminated and contrast of the ultrasound images may be improved accordingly. The proposed research describes the design, synthesis, and evaluation of nanoscale ultrasound contrast agents capable of activating only in the presence of intracellular levels of glutathione (GSH) to determine tumor prognosis noninvasively. We will produce disulfide cross-linked polymer and silica based shells that can be digested by the intracellular GSH levels associated with metastatic disease and potentially drug resistance. Reduction of disulfide crosslinks will soften the shells and allow observation of these contrast agents at a lower imaging power, where great nonlinear signal difference between ON and OFF states of contrast agents is expected. PI Goodwin and Co-Investigator Yildirim will combine their expertise to accomplish this research through completion of the following specific aims: 1. Design and validation of redox-responsive single-layer polymer capsules with sensitivity to GSH; 2. Design and validation of redox-responsive double-layered polymer capsules for longer-term monitoring of cells; and 3. Quantification of GSH in breast cancer cell lines in vitro.